Defects in the biosynthesis of thyroid hormones are among the most common endocrinological problems. However, a systematic and precise definition of these biochemical defects has not been possible because of a lack of adequate knowledge concerning this fundamental biosynthetic mechanism. Hence, our objective is to understand the molecular events involved in the biosynthesis of thyroid hormones, a process which is catalyzed by the integral membrane enzyme, the thyroid peroxidase (TPO). In this application we propose: (1) to study the catalytic, regulatory and structural properties of intact TPO isolated from porcine thyroid membranes, and (2) to apply our biochemical findings to studies of normal and abnormal thyroid tissue. We plan to accomplish this by focusing our biochemical studies on the membrane nature and function of TPO and the mechanism of TPO-catalyzed iodination. Specifically, we propose to: (a) to determine what facets of TPO-catalyzed iodination result from the integram membrane location of TPO by comparing the kinetic and structural properties of intact to fragmented, (trypsin treated) TPO, and (b) to define the chemical nature of its active site by characterizing the TPO-bound iodinating intermediate and its interaction with iodide acceptors, including the thiocarbamide antithyroid drugs. In order to correlate the biochemical findings from our studies on porcine TPO with studies on defects in thyroid hormone biosynthesis, we plan to use thyroid primary monolayer cell cultures, a defined, semi-physiological system, to study: (1) the relationship between TPO activity and thyroid hormone biosynthesis, (2) the synthesis and regulation of TPO activity, at the post-translational and transcriptional levels, by TSH, and (3) the unusual hydrophobic properties of TPO in human thyroid cancer membranes.